JPH0239742B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD This invention relates to the field of liquid chromatography and more particularly to improved methods and apparatus for adding flowable chemicals to chromatographic eluates to increase sensitivity of detection. The use of post-column reactors (reactors located after the chromatographic column) to improve detection in modern liquid chromatographs (HPLC) has long been recognized as a potential, but rarely applied, method. Ta. For example, “Post-column derivatization and digitalis
In the article ``Applications to Glycosides,'' Journal of Chromatography 142 (1977), pp. 271-281, he writes, ``The use of such reaction techniques after column chromatographic separation is similar to classical column techniques. Although it has been known for more than 10 years with modern HPLC (for example, amino acid analyzers), there is almost no mention of modern HPLC.One reason is that HPLC
There are many technical problems that need to be solved." A particular difficulty described in that document is the problem with conventional post-column reactor design. Therefore, “Introduction to Modern Liquid Chromatograph”
In "Modern Liquid Ctromatography", Volume 2 (1979), p. 740, Snyder et al. state that "Reaction detection for modern LC columns primarily requires solving equipment design problems because the use of extra columns is For this reason, reaction detectors have had somewhat limited use in modern LC.''A similar conclusion was made by Frei et al.
al., “Reaction Detectors in HPLC”, Journal
of Chromatographic Science, Vol.17, March
1979, pp. 152-159. In it, the author states, ``certain considerations must be taken to prevent band broadening in the structure of the chromatograph.'' Also Jupille, “UV-Visible Absorption
“Derivatization in Liquid Chromatography”
Journal of Chromatographic Science, Vol.
17, March 1979, pp 160-167, who in other recent publications cited shortcomings in the reactor design as ``the need for hardware modification and the risk of band broadening due to post-column mixing resulting in loss of analysis.'' ”. Moreover, during the metering of the chemicals, fluctuations in the concentration of the chemicals in the effluent occur, and these fluctuations in the concentration of the chemicals adversely affect the spread of the band. This is shown as "noise" in the detector. Generally, metering consistency is preferred, ie, immediate permeation of the drug through the membrane into the effluent. The problem is particularly acute when high concentrations of chemicals are used, since small variations in the meter can significantly disturb the sensitivity of the detector. In the prior art, attempts have been made to use concentrated chemicals to prevent band spread due to sample dilution;
Increase level. Band width is a function of drug/eluate diffusion over time. In previous devices, it took time to mix the chemicals and eluate, and therefore time to spread the sample zone. In the prior art, "hardware modifications" have been attempted to overcome these shortcomings, but this requires additional complex equipment such as air separation methods. It is therefore an object of the present invention to provide an improved liquid chromatographic method and an improved post-column reactor (POST) requiring less hardware modification.
COLUME REACTOR) A particular object of the present invention is to provide an improved method and apparatus for achieving substantially constant pulse-free drug metering and in addition achieving significantly improved diffusion of the drug into the chromatographic column effluent. It is to be. It is another object of the present invention to provide a method and apparatus in which sample dilution is minimized without resorting to the addition of high concentrations of chemicals or resorting to other methods or apparatus. It is yet another object of the present invention to provide a method and apparatus that is sufficiently low cost to use and maintain compared to conventional methods and apparatus. "Hollow fiber membrane" is 20 to 1000 microns,
Preferably has an internal diameter of 50 to 500 microns and is permeable to the flowing drug in contact with the outer wall portion of the fiber (or saturated within the fiber wall matrix) while at least detecting the sample or its derivatives. Refers to a very small tube or fiber that has the property of not allowing liquid chromatography to pass through the membrane, but allowing liquid chromatographic eluate to pass through the membrane through the inner pores of the hollow fiber membrane. A "drug" is a chemical that, when introduced into the chromatographic column eluate through its hollow fiber membrane, chemically reacts directly or indirectly with the target sample species (i.e., it reacts chemically with the target sample species (i.e., the sample species that protects the sample species). means a chemical or combination thereof that reacts directly or indirectly) to enhance the detectability of the sample species or its proportional derivatives as compared to the absence of the hollow fiber membrane/drug combination. "Mobile" refers to a drug that is capable of permeating through the wall or wall portion of the hollow fiber membrane. The object of the invention as stated above consists of a chromatographic column, an introduction member for adding a sample solution to the chromatographic column, and a member for adding an eluent to the chromatographic column, whereby the sample is chromatographically In a liquid chromatographic device, in which a sample component species is eluted through a graph column and its sample components appear in the eluate of the chromatographic column, there is a hollow fiber membrane after the column, said hollow fibers containing a fluid drug. is in contact with
A liquid chromatograph, wherein the drug is permeated through the membrane into the eluate, and the eluate is supplied to a liquid chromatograph detector through the inner pore of the hollow fiber membrane. This is accomplished by a graphing device. Another aspect of the invention includes the steps of: adding a sample solution to a chromatographic column; adding an eluent to the chromatographic column in an amount effective to chromatographically displace the sample species from the chromatographic column; A method of analyzing a sample by liquid chromatography comprising a step whereby the chromatographically displaced sample species ultimately appears in the eluate of the chromatographic column, in which the eluate from the chromatograph is The hollow fiber membrane is used to direct the fluid drug into the eluate of the chromatographic column through the inner pore of the hollow fiber membrane, ultimately feeding into a liquid chromatography detector, and prior to detection. This method is characterized by increasing the sensitivity of detection after permeation. An essential feature of the invention is the use of single or multiple hollow fibers in conventional chromatographs. In a preferred device, a number of hollow fibers are embedded in the tubesheet for the purpose of separating the inner bores or inner portions of the fibers. The liquid chromatography device then has means for connecting the inner portions of the fibers. The central portion of the hollow fiber is immersed in a fluid chemical. The hollow fiber device can be easily installed into standard or commercially available chromatography equipment with minimal hardware changes. Hollow fiber membranes useful in the present invention may be porous in nature in the material from which the fibers are made. However, not all materials from which fibers are made are porous.
For example, polyethylene membranes are not inherently porous. However, polyethylene membranes react with chlorinated sulfonic acid and become porous to ions. The invention features a hollow fiber membrane structure. The present invention is not an invention of the hollow fiber membrane structure itself. Commercially available hollow fiber membranes developed for other purposes can be used in the methods and apparatus of the present invention. In addition to its ability to contact and permeate chemicals and resist migration of at least a portion of its sample, a desirable performance of the hollow fiber membrane is its resistance to contact with the various liquids to which it is exposed. Porous cellulose membranes made by the method of US Pat. No. 3,546,209 are particularly useful in this regard. Such membranes may be isotropic or anisotropic in structure. The permeation property is essentially a size-selective property and is widely applied to permeate a wide variety of different drug classes. Synthetic polymeric membranes typically made from polyolefins and also silicone rubbers as well as other possible polymeric materials are suitable for use in the present invention. For example, polyethylene membranes react with chlorinated sulfonic acid to transform polyethylene sulfonate membranes. Polyethylene sulfonate membranes are negatively charged. This is because the sulfonate film on the film has a negative charge. Ions with charges similar to those of the polyethylene sulfonate membrane, such as chloride ions, tend to be removed from the membrane. This is because two or more similar charges repel each other. This phenomenon is called the "Donnan effect." On the other hand, ions having an opposite charge to the polyethylene sulfonate membrane, such as sodium ions, are not excluded from the membrane. That is, polyethylene sulfonate membranes tend to allow cations to readily permeate across the membrane. However, it tends to reject the permeation of anions across the membrane. That is, polyethylene sulfonate membranes are selective in their permeation. This is because the membrane is more permeable to cations than anions. The "Donnan effect" is different from selectivity. However, the selectivity can be explained by the "Donnan effect". When an ion exchange membrane with a negative charge is used,
Due to the Donnan effect, cations can more easily pass through the membrane than anions. On the other hand, when non-ionic membranes are used, such as silicone rubber membranes, the "Donnan effect" is not a factor in the selectivity of the permeable substances. Ion exchange membranes are used, but when the substance to be permeated through the membrane is not an ion, the "Donnan effect" is also not a factor in selectivity. "For example, ion-exchange membranes pass water faster than petroleum distillates. This is because ion-exchange membranes have higher selectivity for water than petroleum distillates. However, this principle has nothing to do with the Donnan effect." It is. Silicone rubber membranes (non-ionic membranes usually do not allow ionic substances to pass through; ion-exchange membranes usually do not allow non-polar substances (e.g. petroleum distillates) to pass through; microporous membranes do not allow all that physically passes through their pores. The "leakage" property of the hollow fiber membranes used in the present invention is not a necessary requirement; for example, leakage of components other than the sample through the fiber walls (although often not unreasonable) would make the analysis unreasonable. i.e. the eluents used in the present invention are generally characterized as non-interfering or substantially non-interfering; therefore, solvent leaking through the membrane in either direction will not dilute or interfere with the sample. In addition, although it is desirable to have a hollow fiber membrane that completely rejects sample permeation, this is neither an absolute nor a practical requirement. Excessive sample loss does not necessarily mean significant adverse consequences.
A 1000-fold increase in sensitivity can be achieved when the chemical is introduced into the effluent from the column prior to detection.
This increase in sensitivity is considerably greater than the loss in power due to leakage of 90% of the sample. That is, the column prevents significant leakage of the sample through the membrane into the chemical compared to the permeation of the chemical through the membrane into the effluent by using a specific membrane. can. In this case the sensitivity of detection of sample components is increased. If multiple hollow fibers are used to minimize zonal spread due to the geometric flow pattern factor, each creates the same resistance to flow and thus the outflow is split and individual It is desirable that the flow occur at a uniform velocity as it flows through the multiple passages of the fiber. That is, the residence time of the flow rate in the individual fibers is essentially constant and the tendency for band broadening is greatly reduced. This is typically accomplished by using multiple fibers of the same inner diameter and length. Obviously to completely overcome that effect,
The fibers are matched to produce exactly equal flow characteristics. Also, in general, the larger the fiber, the greater the tendency to cause band widening due to inherent fluid tension at the fiber wall/flow interface, so that if it is sufficiently noticeable or extended enough, , causing analysis loss. The effect is extremely small allowing the drug to pass faster by means of large surface areas, often allowing minimal band broadening based on the use of short transport paths and the diffusion of sample bands from each other as a factor of time. By using a large number of fibers the diameter is minimized. The diffusion rate of the drug in the chromatographic eluate is effectively improved by using multiple hollow fiber membranes of small diameter. That is, sufficient advantages in the required parameters are achieved through the use of a post-column reactor design based on the use of a large number of hollow fibers with sample permeation rejection properties, which fibers are connected between the chromatographic column and the detector. are connected parallel to. Fibers having an inner diameter of 100 to 300 micrometers for use in conventional liquid chromatographs are particularly suitable for use in multiple fiber devices. Scott et al.
Journal of Chromatography, Vol.169 P51
(1979), extremely small fibers can be used, around the usual manufacturing limits of about 20 μm internal diameter. Its fiber wall thickness is
3808267, the relationship between film thickness and permeation rate becomes irrelevant if the materials are different. Therefore, in the present invention, the thickness of the fiber is not a necessary requirement. Generally, the wall thickness of the fibers as used in the present invention may be from 5 to 250 μm. The length of the fiber also varies depending on the permeability and/or rate of diffusion through the fiber wall. It is generally desirable to use fibers with a length of 10 to 200 cm. The limitations on length (and minimum internal diameter) are ultimately a function of the pressure drop across the fiber and the backpressure on the fiber. Too much backpressure will destroy the fiber, ie, limit the pump pressure. This is avoided by equalizing the internal pressure and the pressure surrounding the fiber, such as by holding the fiber in a pressurized vessel. To take that latter precaution, the fiber length can increase the pressure to the point where the chromatographic displacement species analysis is not adversely affected. The fiber, or a portion thereof, contacts the flowable drug and permeates the drug. The temperature, the pressure difference with the eluent and the concentration of the drug significantly influence the permeation rate. The drug consists of a pure liquid, a pure gas, or a solution of the drug in a diluent or carrier, so that the drug is fluid and passes through the fiber wall. In some cases, an inert diluent such as methylene chloride is used to diffuse into the membrane and increase the rate of permeation of the drug through the membrane. Water and other types of liquids such as acetonitrile are typically used as drug diluents. This embodiment can be used, for example, with ion exchange hollow fiber membranes containing the ions to be exchanged,
The drug ions are thereby exchanged with the exchangeable ions of the membrane and thus ultimately diffuse into the chromatographic column eluate. In some cases, the permeation of opposite ions of the drug through the ion exchange membrane interferes with or is detrimental to the analysis. The "Donnan effect" theoretically prevents the permeation of a drug's opposite ion through an ion exchange membrane. Two types of drug reservoirs or containers are particularly used. The preferred form is generally described as a static reservoir, although the drug can be agitated so that no concentration differences occur. Alternatively, fresh drug can be continuously pumped into contact with the fiber. The latter embodiment is advantageous if contamination of the reservoir by leakage of chromatographic eluate would have a detrimental effect on the analysis. In such embodiments, the fibers are placed, eg, coaxially, within a preferred flexible container to define an annular space between the fibers and the inner wall of the tube. Stationary reservoirs are preferred because of their reduced complexity and typically very comparable effectiveness to dynamic reservoir embodiments. The reaction kinetics of the chemical addition method as well as conventional chemicals are considered background to the present invention. That is, the special chemicals and reactions developed for detection purposes and used in the present invention are distinct from the prior art. A detailed description of reactions considered suitable for use in the post-column eluate reaction method can be found in Snyder et al., Supra 740:746.
Page and Gfeller et al., Frei et al., and
Similar techniques are given in previous citations by Jupille, and Vance Nau et al.,
“Application of Microporous Membrances to
Chemiluminescence”，Analytical Chemistry，
Also given in Vol.51, No.3, March 1979, pp424-428. The favorable reaction conditions necessary to promote the reaction must of course be present in this invention along with other chemical addition conditions. That is, reactions requiring large amounts of water in the final reaction mixture generally require the use of aqueous eluents. That latter requirement is generally suitable for ion-exchange and reverse-phase liquid chromatographic separation processes, or similar liquid chromatographic processes that use aqueous eluents or fluid phases with which the chemicals are at least partially miscible. For liquid chromatographic techniques, as well as fractional common phase chromatographic techniques, the present invention typically applies to the selection of drugs that require an organic solution with which the drug is at least partially miscible. Limited. Liquid chromatographic detectors useful in the practice of the present invention are particularly advantageously photometers, spectrophotometers, and fluorometers, which alter or cause the optical absorption of a sample species in its chromatographic eluate. or with chemicals that cause the formation of fluorescent derivative products. Other liquid chromatographic detectors which are advantageously used in the present invention are, for example, differential refractometers, electrochemical detectors, radioactive detectors and conductivity detectors. Additional objects and devices of the invention will be explained in detail with reference to the accompanying drawings. Referring to FIG. 1, there is shown a chromatographic column 10 comprising a housing containing separation means in the form of a particulate packing or gel which is eluted to separate a sample solution into component species. Various types of separation means can be used to construct suitable chromatographic columns as described by Snyder et al. A preferred means for adding an eluent solution or a flowable liquid to the chromatographic column 10 comprises an eluent reservoir 12 containing an eluent solution 14, which solution is pumped into a pump 16 equipped with a tube 18 having elastic walls. It is then removed from its storage 12. The preferred means for adding the sample is a sample injection valve 2 which can be equipped with a syringe.
Consists of 0. Samples added to the system at valve 20 are expelled from the apparatus by pumping eluent solution through optional guard column 22 and into chromatographic column 10. The sample is eluted through column 10 and its component species are ultimately chromatographically displaced into the chromatographic column eluate, which eluate is subjected to drug loading means as detailed below. That is, it is sent to the post-column reactor 24. The chemical addition means or reactor is optionally provided with a coil or tube (not shown) between the reactor 24 and the detector 26. The reason for providing this coil or tube is as follows. The chemical reacts with the sample to form some derivative. If the rate of reaction between the drug and the sample is slow,
It is possible that the reaction between the drug and the sample is not complete by the time the derivative reaches the detector. To solve this problem, the length of the coil or tube between the reactor 24 and the detector 26 is made long enough to complete the reaction until the derivative reaches the detector. Optionally, the delay coil is immersed in the chemical solution and/or the delay coil and/or suitable temperature control means (not shown) are provided, such as a suitable temperature control plate for heating the solution or fluid that heats the chemical solution. The temperature is maintained at a controlled temperature by Next to the delay coil is a detector 26 of a type suitable for liquid chromatography. In the detector, the eluate has a light absorbance,
An electrical signal is generated proportional to the monitored characteristic, such as fluorescence, and the signal is sent from the detector to a recorder 28, such as a recording strip, or to an integrator. Such detectors are known in the art. Referring to FIG. 2, the preferred type of drug addition device is a static storage design, which consists of a reservoir or drug container 30, preferably made of glass or a similar inert material. A fluid drug 32 is contained in the reservoir. The drug is filled spontaneously through a screw cap or other suitable closure 34. The hollow fiber 36 is connected to the eluate inlet supply section 3
8 and the eluate outlet supply section 40 in the drug. The connection between the hollow fiber and the inlet supply and the outlet supply is made by providing an opening 42 in the closure 34.
Each opening is provided by the use of a general purpose epoxy resin adhesive as shown in FIG. A tube 48 for supplying chromatographic column eluate is connected to a threaded nipple 50 on adapter 44 using a standard connection net 52 and ferrule 54. The eluate outlet supply is also connected by a tube 56 to the detector 26 through a similar connecting net 52 and ferrule 54. A preferred type of securing the end of a hollow fiber into the female portion of the adapter 44 is to mold fitting 58 around the end 60 of the fiber. This fitting should be replicated as a form for the mold using Silastic mold-forming rubber J-RTV to make the mold using a commercially available fitting.
made by using rubber compounds such as When epoxy resin is used to secure the ends of the fibers, the fibers are screwed into the mold by drilling small diameter holes in the fibers for threading purposes. Dow 331
An epoxy resin, such as Epoxy Resin/Ancamine LT Hardener, is poured into the mold and cured with the fibers, which are wet or dry depending on the end use. After removal from the mold, the fibers are adjusted as necessary. A fiber coating layer 62, such as Silastic 730 RTV fluorinated silicone sealant, is preferably applied to the area adjacent to the fit to avoid stress in the area adjacent to the fit, and thus reduces the physical properties of the fiber. minimize damage that may occur during handling. The epoxy resin system is useful with aqueous chemical and eluent systems, certain aromatic solvents and certain hydrocarbons. For chemical solutions or eluents that chemically attack epoxy, fittings 58 manufactured similarly to epoxy fittings.
Silastic Brand J-RTV is used effectively for a long time. The fitting 58 is also suitably manufactured using a hollow commercially available fitting with the fibers passing through it;
Filled with a sealant material such as 730RTV Sealant and then cured. The fitting that specifically explains this is, for example, about 2
It is a tube-like material approximately 1 inch (2.54 cm) long with an inner diameter of 1.5 mm. The outer diameter of the fitting is, for example, approximately 1/4 inch (6.4 mm). There are 28 streaks per inch (2.54 cm) inside. The hollow fiber is forced into the 2 mm inner diameter of the fitting. A silicone rubber sealant or epoxy resin is injected between the fitting and the hollow fiber. A preferred embodiment of a drug addition device having a fiber immersed in a counter-flowing flowable drug is shown in FIG. In this device, a fiber or bundle of fibers inside a tube is inserted into the stainless steel interior by pulling the fibers using water as a lubricant. T-shaped fittings 70 are each secured to opposite ends of the jacket 68 using ferrules 74 and nuts 72 to form attachments. The exposed portion of the hollow fiber (outside of each T) is dried and inserted into a sealing tube 76, preferably made of stainless steel. Approximately 6 inches of fiber is exposed between the sealing tube and the T-70;
The exposed fibers are then coated with a suitable sealant, such as Silastek 732 RTV silicone rubber, as shown at area 66. The sealing tube is then joined to the T using tube nut 78 and ferrule 80. Additional RTV sealant is 20
A hypodermic tube with a gauge needle is used to introduce the sealing tubes, completely filling each sealing tube, but being careful not to get excess rubber into the T. Ion exchange hollow fibers typically swell when wetted with water. If the ion exchange hollow fiber is to be used with water-based effluents or water-based chemicals, it must be moistened with water during curing of the sealant. A razor blade is used to cut a single section of the fiber so that it lies flat at the end of the sealing tube. The device is a reduction union assembly 8
2 is coupled to the apparatus of FIG. A reduction union is a tube fitting for connecting two tubes of different diameters together. The reduction union assembly (28 in FIG. 3) consists of a central tube, a large tube nut, a large ferrule, a small tube nut, and a small ferrule. The tube is drilled from one end to the other. One end of the tube is threaded to accept a large nut and large ferrule, and the other end of the tube is threaded to accept a small nut and small ferrule. 82 in Figure 3
and 94 is a reduction union assembly. Similarly, a reservoir and pump (not shown) are connected to tee 70 to supply the drug through tubes 84 and 86. These latter connections are from tube elements 88 which are coupled at one end to each tee 70 through a tube nut 90 and ferrule 92 and to drug inlets and outlets 84, 86, respectively, through a reduction union assembly 94 at the opposite end. Become. The assembled device defines a catalytic flow path consisting of the collecting holes of the hollow fibers and the outer space of the fibers in jacket 68, which connects the chromatographic column and the chemical source (not shown). The described drug addition device operates by receiving eluate from a chromatographic column through the interior of the hollow fiber. At the same time, the fluid drug solution (static or dynamic) flows onto the outer surface of the hollow fiber, so that the drug permeates into the chromatographic eluate. The invention is illustrated by the following examples. EXAMPLE 1 Hollow Fiber Membranes A variety of hollow fiber membranes are used in the manufacture of the reagent addition device used in the present invention using the preferred static reservoir device. These are: Contrived product (A) (Prior to sulfonation) Constructed from a hollow fiber with an outer diameter of 380 μm and an inner diameter of 300 μm, the contrived product (A) is produced by extruding it from a spinneret using a well-known method. do. The fiber is made from low density polyethylene, manufacturing code number 4005, which is utilized on a commercial scale by the Dow Chemical Company. For sulfonation purposes, the fibers are wound onto glass cylinders (frameworks) and fixed with Teflon tape. It is desirable that the portion of the fiber to be connected not be sulfonated, since sulfonation weakens the fiber. This is accomplished by creating a separate section looping at a 90° angle to the circumferentially wrapped fiber section to form a loop offset from the cylindrical end. Using a Teflon cord attached to the loop, the fiber is suspended in a three-necked flask equipped with a concentrating and heating cylinder. Add sufficient volume of a 10% (v/v) chlorosulfonic acid solution in methylene chloride to the flask, submerge the fiber (not the end of the roux), heat at reflux conditions (42°C), and incubate the sulfonic acid for approximately 30 minutes. become The fibers are then collected, placed in methylene chloride, soaked for 30 minutes, and then washed with deionized water. The loop is cut to provide a non-sulfonated end for injection with fixture 58. Capacity is approx.
1meq/gm. A device constructed in this manner preferably uses seven sulfonated fiber threads (each 8 inches long). Innovative product (B) Product name from Bio Rad Research Institute
Outer diameter 300μm obtained as SR-α/751-7010,
The device (B) is constructed by using four hollow fiber threads with an inner diameter of 230 μm and a length of 8 inches. This fiber is 40% α-methylstyrene/
It is believed to consist of a copolymer of 60% polymethylsiloxane. Convenience (C) before being manufactured by using 16 fiber threads each 8 inches long with dimensions of an outer diameter of about 375 μm and an inner diameter of about 300 μm with a capacity of about 0.5 meq/gm. From the basic sulfonation procedure and using the material from Devise (A), Devise (C) is produced. Device (D) Using 9 fiber threads, each approximately 10 inches long, this device uses the same fibers as device (A), but has been sulfonated to a length of approximately 10 inches.
A capacity of 7meq/gm was achieved. Improved product (E) The improved product is Svectrum Medical Industries, serial number 132272, registered trademark SPECTRA/
Manufactured from microporous cellulose hollow fibers obtained as a product sold as POR Hollow Fiber (HF) membrane. This device is
It is described as having a molecular weight in the range of 500 to 2000,
It consists of 10 fiber threads, each 6 inches long. Example 2 Nitrophenol Detection Separation of a wide variety of nitrophenols by using a silica-based column is amenable to acidic eluent PH conditions (pH approximately 6) due to component degradation and/or column PH limitation reasons. is necessary.
While this nitrophenol absorbs at a wavelength of 280 μm, interfering organic substances in the sample matrix exhibit only a small detection sensitivity. The low detection sensitivity due to interfering organic substances can be eliminated by changing the PH of the column effluent using the present invention. The PH of the column effluent can be changed by _NH3 . By changing the PH of the column effluent, we create induced samples that are sensitive to the detector at different wavelengths. This experiment used the devised product (A) and 30
% ammonium hydroxide aqueous solution. This experiment is carried out under the following conditions: 12 (v/
v)% methanol aqueous eluent, 0.08M sodium perchlorate, 0.04M ammonium acetate PH with glacial acetic acid
6.1 and pumped with 100 μl sample at 1.8 ml/min using a mieton Roy mini pump through a Ryeodyn 7120 sample injector valve. This sample consisted of an identified organic matrix containing significant nitrophenol, Watmann Partecil Sax.
Added to 10/25 chromatographic column, LDC
Detect using a 1203 (Laboratory Data Control) photometer with a 410 nm detection wavelength. The Spectrophis IX SP-4100 is an integrator used to record and calculate the output signal. Typical nitrophenols detected by this system are 2,4,6-trinitrophenol, 2-sec-butyl-4,6-dinitrophenol, 3-sec-butyl-2-hydroxy-5-nitrobenzenesulfone. acid and 3-sec-butyl-4-hydroxy-5-nitrobenzenesulfonic acid. The detection limits for these compounds are below ppm. At this detection wavelength and PH change, this system turns out to be very selective. In an experiment similar to the above, important 2, 4, 6
-Trinitrophenols similarly elute 2-
Detected as a mixture with sec-butyl-4,6-dinitrophenol. In this case, a 2M aqueous hydrochloric acid solution is used as the reagent. Chromatographic eluents are limited to a pH of about 2. The dinitrophenol peak can be selectively narrowed by adjusting the pH. It is then decomposed to detect important trinitrophenols in the ppm or less range. Example 3 Fluorescamine Addition The fluorescamine reaction is a particularly important reagent addition reaction used, for example, in the detection of primary amines. This experiment illustrates a specific example of a fluorescamine reaction used in accordance with the present invention. This experiment is performed using a 2 mg/fluorescamine solution in acetonitrile as the reagent addition device, device (B), and the reagent to soak the fiber. 1 ml/30 (v/v)% acetonitrile in an eluent aqueous solution containing 0.01 M ammonium acetate.
Uses a Varian 8500 chromatographic pump to pump in minutes. Rheodyne 7120 inlet (20 μ 1 loop) is used for Waters μ-C-18 analysis of standard sample (1 ppm α-aminotoluene aqueous solution).
Used to add to HPLC column. The sample is detected using a DuPont 836 fluorescence detector operated to excite and emit at 390 nm and 475 nm, respectively. Strong peak (off scale)
is obtained at ppm sample concentration levels. Example 4 Ninhydrin Reaction The ninhydrin reaction is an excellent tool for the detection of amino acids and is commonly used by commercial amino acid analysts, producing blue-colored derivatives that can be detected with a visible light photometer. This reaction can be advantageously used in accordance with the present invention as illustrated in this example. Under the conditions of this experiment, an eluent of 0.02 M citric acid aqueous solution adjusted to pH 4.0 with aqueous sodium hydroxide solution was pumped through a Rheodyne 7120 injection valve with a 200 μl loop at 0.52 ml/ml with a Milton Leumini pump.
Pump for 1 minute and then periodically inject 50 ppm glycine to simulate peak drop in the separation column. The devised product (C) is used for the purpose of adding a reagent, and 100 ml of a 10% ninhydrin aqueous solution is used as the reagent. A hot plate is used to maintain the reagents at 90°C ± 5°C. The ninhydrin-treated solution is then passed through a heating delay coil (2 m, coiled Teflon tube maintained at 90°C), which delays the reaction from occurring for 3 minutes. The reaction eluate is
Perkin-Elmer LC operating at 600nm
Monitored using a 55UV/VIS absorption photometer. The blue absorption peak due to the ninhydrin and amino acid reaction product is recorded on a Sergeant Welch model SG recorder. Its prominent peak shows a detection limit at ppm level. It is estimated that approximately 75% of the amino acids reacted under the conditions used. During the process of continuous sample injection, no decolorization of the ninhydrin reagent solution is observed. Example 5 Iodine Reaction Peroxide and other relatively strong oxidizing agents form 1 ₃ ^- which is the oxidation of 1 ^- to 1 ₂ when there is an excess of 1 . This reaction is useful, for example, in determining the presence of peroxides or other strong oxidizing agents in industrial process streams and products. As an example of utilizing this reaction in the present invention, 100
mg/(NH ₄ ) ₂ M ₀ O ₄ aqueous eluent, PH5 54
℃±0.2

[Table] The detection limit calculated from this data is approximately
Estimated to be 0.1ppm. This method is thought to show a linear relationship up to about 50 ppm of hydrogen peroxide. Example 6 Cellulose Fiber The iodine reaction is similarly applied to detect species such as NO ₂ ^- , ClO ₃ ^- , bleach and Cl ₂ . This example illustrates the detection of sodium chainite using cellulose hollow fiber engineering (E) and 0.1N aqueous potassium iodide as the reagent. To demonstrate the potential of the reactor-detector combination, a standard sample from 100 to 1000 ppm, a 20 μ sample injection size, was injected into eluent water flowing at 1 ml/min and passed through the device (E). The eluate of the device was monitored by a Perkin-Elmer LC-55 photometer set at 360 nm. Although not efficient, an excellent detection sensitivity response was achieved, and a detection sensitivity comparable to that of hydrogen peroxide in the previous example was achieved.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an apparatus for liquid chromatography using post-column chemical addition according to the principles of the present invention.
FIG. 2 is an enlarged sectional view showing details of the post-column reactor used in the apparatus of FIG. 1, and FIG. 3 is a cross-sectional view of a reactor similar to FIG. 2 showing a modified version of the post-column reactor. .

Claims (1)

Claims: 1. Adding a sample solution to a chromatographic column; adding an amount of eluent to the chromatographic column effective to chromatographically displace the sample from the chromatographic column;
The thereby chromatographically displaced sample ultimately appears in the effluent of the chromatographic column, passing the effluent from the chromatograph through the inner pores of a hollow fiber membrane, in between which the fluid drug is By means of a liquid chromatographic column consisting of using a hollow fiber membrane to permeate into the effluent and finally introducing the effluent into a detector whose detection sensitivity has been enhanced by the introduction of the drug. How to analyze the sample. 2. A method according to claim 1, characterized in that it uses a plurality of hollow fiber membranes connected between the chromatographic column and the detector and immersed in a fluid chemical. 3. The method of claim 1, wherein the fluid drug is contained in a non-continuously refilled reservoir. 4 a chromatographic column, an injector for applying a sample solution to the chromatographic column, a member for applying an eluent to the chromatographic column, whereby the sample is eluted through the chromatographic column and the sample is A device for analyzing samples by liquid chromatography, which consists of appearing in the effluent of a chromatographic column, in which a hollow fiber membrane is present after the column, said hollow fibers being in contact with a fluid drug;
A liquid chromatograph, wherein the drug is permeated through the membrane into the effluent, and the effluent is supplied to a liquid chromatograph detector through the inner pore of the hollow fiber membrane. A device that analyzes samples graphically. 5. Claim 4, wherein the reservoir containing the fluid drug is replenished with the drug discontinuously.
Apparatus described in section.